1. Field of the Invention
This invention relates to a liquid spotting method and a liquid spotting device for spotting a sample liquid such as blood or urine, a diluent liquid, a reference liquid or the like onto a member to be spotted with the liquid such as a dry chemical analysis element, a dilution cup or the like, and more particularly to such a liquid spotting method and a liquid spotting device in which a liquid held in a disposable spotting tip is continuously spotted onto a member in a constant amount.
2. Description of the Related Art
There has been put into practice a dry ("dry-to-the-touch") chemical analysis element with which a specific chemical component or a solid component contained in a sample liquid can be quantitatively analyzed by only spotting a droplet of the sample liquid onto the element. See, for instance, U.S. Pat. Nos. 3,992,158 and 4,292,272. When such a dry chemical analysis element is used, the sample liquid can be analyzed more easily and more quickly than when the conventional wet analysis method is used, and accordingly the dry chemical analysis element is very convenient for medical facilities, laboratories and the like where lots of sample liquids have to be analyzed.
When quantitatively analyzing the chemical components or the like contained in a sample liquid using such a dry chemical analysis element, a droplet of the sample liquid is spotted onto the element and is held at a constant temperature for a predetermined time (incubation) in an incubator so that coloring reaction occurs, and the optical density of the color formed by the coloring reaction is optically measured. That is, measuring light containing a wavelength which is pre-selected according to the combination of the component to be analyzed and the reagent contained in the reagent layer of the element is projected onto the element and the optical density of the element is measured. Then the concentration of the component to be analyzed is determined on the basis of the optical density using a calibration curve which represents the relation between the concentration of the biochemical component and the optical density. Thereafter the chemical analysis element is taken out from the incubator and discarded in a discarding box.
The chemical analysis element generally comprises a chemical analysis film in the form of a chip having a reagent layer formed on a support sheet of organic polymer or the like. The chemical analysis film chip is sometimes used as it is, and sometimes used in the form of a slide comprising such a chemical analysis film chip and a frame of organic polymer or the like which supports flat the film chip. In such a chemical analysis element, it is important that the sample liquid is spotted onto the element accurately in a predetermined amount in order to ensure accuracy of the measurement.
Further when a sample liquid is diluted to a predetermined concentration, the sample liquid and diluent must be spotted in a dilution cup accurately in predetermined amounts.
Further there has been known a dry electrolyte analysis slide for measuring the concentration of an electrolyte in a sample liquid. The electrolyte analysis slide has a pair of electrodes. When measuring the concentration of an electrolyte in a sample liquid using the electrolyte analysis slide, a sample liquid and a reference liquid are spotted onto the slide and the concentration of the electrolyte is determined on the basis of a potential difference. Also in this case, the sample liquid and the reference liquid must be spotted accurately in predetermined amounts.
As a way of spotting a liquid such as a sample liquid, diluent liquid, reference liquid or the like onto a member such as a dry chemical analysis element, a dilution cup or the like, there has been known a method in which the liquid is once sucked in a suction nozzle and is discharged on the member with the suction nozzle washed every time the kind of the liquid is changed. However this method is disadvantageous in that when the liquid remains on the nozzle, contamination occurs to deteriorate accuracy of measurement and that the nozzle washing process deteriorates the time efficiency of the system. In order to overcome such a problem, there has been put into practice a method in which a disposable spotting tip is mounted on said suction nozzle so that the liquid is sucked in the spotting tip and the spotting tip is changed every time the kind of the liquid is changed. See U.S. Pat. No. 4,340,390 and the like.
When spotting is effected by sucking liquid in an amount corresponding to one spotting in the spotting tip and discharging the whole liquid in the spotting tip onto a member to be spotted with the liquid, the liquid cannot be spotted accurately in a predetermined amount. That is, a part of the liquid is apt to remain on the end of the spotting tip under a surface tension and an attempt to entirely spot the liquid held in the spotting tip results in forming bubbles in the spotted liquid.
It may be expected that the liquid can be spotted more accurately in a predetermined amount when the liquid is sucked in the spotting tip in an amount larger than that for one spotting so that only a part of the liquid held in the spotting tip is discharged. Further when a liquid is to be spotted onto a plurality of members to be spotted with the liquid, the steps of sucking the liquid in the spotting tip in an amount larger than that for one spotting and discharging a part of the liquid held in the spotting tip onto the member are repeated for each of the members or the liquid is sucked in the spotting tip in an amount larger than the number of spottings times the amount for one spotting and discharged onto the members in sequence by the amount for one spotting.
In the method disclosed in the above identified United States patent, the suction system is vented to atmosphere each time the liquid is sucked into the spotting tip to return the pressure inside the spotting tip to atmospheric pressure so that the liquid level in the spotting tip is lowered to a position where the liquid in the spotting tip balances with surface tension on the inner surface of the spotting tip and then the liquid in the spotting tip is discharged.
However, when the liquid is sucked in the spotting tip in an amount larger than the number of spottings times the amount for one spotting and discharged onto the members in sequence by the amount for one spotting, the amount of the liquid actually spotted fluctuates from one spotting to another.
That is, as shown in FIG. 11 where a result of an experiment is shown, the amount of the liquid spotted in the first spotting is larger than that spotted in any other spotting. In this experiment, a new spotting tip was mounted on the suction nozzle and water was sucked in the spotting tip in an amount larger than that for a predetermined number of spottings at one time. Then the water was discharged onto the predetermined number of chemical analysis elements at intervals of 9 seconds by introducing the same discharge pressure into the spotting tip for each spotting so that a target amount of (e.g., 10 .mu.L) water is spotted in each spotting. Three kinds of chemical analysis elements were used and the amount of spotted water differed by the kind of the chemical analysis element. However, irrespective of the kind of the chemical analysis element, the actual amount of water spotted in the first spotting was larger than that spotted in any other spotting by about 0.7 to 0.9 .mu.L.
One of the three kinds of chemical analysis element was for measuring glutamic-pyruvic transaminase (GPT-P), another for measuring lactate dehyrogenase (LDH-P) and the other for measuring creatinine. The experiment was carried out in an atmosphere at a temperature of 32.degree. C. and a relative humidity of 30%.
It has been found that fluctuation in the vapor pressure in the spotting tip causes the actual amount of water spotted in the first spotting to be larger than that spotted in any other spotting. That is, when water is sucked in a new spotting tip, which is dry, the space in the spotting tip above the water is at a vapor pressure at the temperature and the humidity of the atmosphere as shown in FIG. 12A. (In this state, after water is sucked in the spotting tip, air is sucked in the spotting tip to raise the water level L.) However as the time lapses, the water in the spotting tip evaporates and the pressure inside the spotting tip rises to lower the water level L as shown in FIG. 12B. Accordingly when a predetermined discharge pressure is introduced into the spotting tip to increase the pressure inside the spotting tip to discharge the water as shown in FIG. 12C, the pressure inside the spotting tip is excessively increased by a value corresponding to increase in the vapor pressure and accordingly an excessive amount of water is discharged in the first spotting. After the first spotting, the vapor pressure inside the spotting tip is saturated and does not fluctuate, whereby the amount of spotted water is stabilized.
The reason why the amount of spotted water differs by the kind of chemical analysis element may be the difference in wetting due to difference in the reagent layer between the chemical analysis elements.
When the inside of the spotting tip is vent to atmosphere for each spotting as in the above identified United States patent, generation of difference in amount of spotted liquid due to influence of the vapor pressure may be prevented. However since the position where the liquid in the spotting tip balances with surface tension on the inner surface of the spotting tip varies according to the wetting (water repellency) of the inner surface of the spotting tip, the viscosity of the liquid and the like, the amount of spotted liquid can fluctuate. Further the method is disadvantageous in that an off-off valve is required in the suction system, which complicates the system.
Thus in continuously spotting liquid onto a plurality of members to be spotted with the liquid using a disposable spotting tip, there has been a problem that there is a difference in the amount of liquid actually spotted onto the member for a given discharge pressure between a spotting before the vapor pressure is saturated and a spotting after the vapor pressure is saturated.
Further as the method of spotting a sample liquid onto a dry chemical analysis element, there has been known a method in which after the sample liquid is sucked into a disposable spotting tip, a droplet is formed on the lower end of the spotting tip, the spotting tip is moved downward toward the chemical analysis element until the droplet is brought into contact with the chemical analysis element and the spotting tip is stopped to permit the droplet to spread over the upper surface of the chemical analysis element. Further as disclosed in U.S. Pat. No. 4,340,390, there has been known a method in which the spotting tip is lowered to a position where the lower end thereof is at a predetermined distance from the upper surface of the chemical analysis element and then the sample liquid in the spotting tip is discharged at a predetermined rate.
With either of the methods, even a slight coagulation of the droplet on the lower end of the spotting tip can cause error in the amount of spotted sample liquid when a very small amount (e.g., a few .mu.L) of sample liquid is to be spotted.
In order to suppress generation of the error due to coagulation of the droplet, it has been proposed to suck air into the spotting tip after sucking the sample liquid, thereby raising the lower surface of the sample liquid in the spotting tip above the lower end of the spotting tip as disclosed in Japanese Unexamined Patent Publication 1(1989)-184464. However it has been found that this method is disadvantageous in that a bubble is formed in the lower surface of the sample liquid in the spotting tip, which can cause error in the amount of spotted sample liquid.
That is, generally the suction nozzle on which the spotting tip is mounted is supported by an arm member and the arm member is moved up and down and in horizontal directions in order to spot the sample liquid held in the spotting tip onto a chemical analysis element. It has been found that during such motions of the arm member, the spotting tip 301 is vibrated and the vibration of the spotting tip 301 generates a bubble 305 as shown in FIG. 13B in the lower surface of the sample liquid 304 which has been sucked inside the spotting tip 301 so that the lower surface thereof is positioned higher than the lower end of the spotting tip 301 as shown in FIG. 13A.
This inventor has recognized that the bubble 305 is formed in the following manner. That is, the spotting tip for spotting a very small amount of liquid should be very small in inner diameter. However when the entire spotting tip is thin, the spotting tip becomes apt to deform and apt to swing in the lateral direction to a large extent by mechanical vibrations, which prevents an accurate spotting action. Accordingly it is preferred that the spotting tip be thick as a whole to increase strength in deform with the lower end portion made very thin to enable spotting of a very small amount of liquid. Thus the spotting tip for spotting a very small amount of liquid naturally should comprise an upper portion 301a (FIG. 13B) having a larger diameter, a lower portion 301b having a smaller diameter and a tapered portion 301c connecting the upper and lower portions 301a and 301b. That is, the inner diameter of the spotting tip discontinuously increases toward the direction away from the sucking port of the spotting tip in the range where the sucked liquid occupies. With the spotting tip 301 of such a shape, when the sample liquid 304 is sucked into the spotting tip 301 to a position where the lower surface of the sample liquid 304 is higher than the boundary 301d between the lower portion 301b and the tapered portion 301c, thin film of the sample liquid 304 formed on the inner surface of the spotting tip 301 when the sample liquid 304 is sucked upward frequently comes to extend across the inner space of the spotting tip 301 at the lower end of the thin film due to vibrations, thereby forming a bubble about the tapered portion 301c.
Thus there has been a demand for a liquid spotting method which can spot a very small amount of liquid with a spotting tip having a tapered portion without formation of a bubble in the lower portion of the liquid sucked into the spotting tip above the lower end thereof.